Modeling the Orthosteric Binding Site of the G Protein-Coupled Odorant Receptor OR5K1.

With approximately 400 encoding genes in humans, odorant receptors (ORs) are the largest subfamily of class A G protein-coupled receptors (GPCRs). Despite its high relevance and representation, the odorant-GPCRome is structurally poorly characterized: no experimental structures are available, and the low sequence identity of ORs to experimentally solved GPCRs is a significant challenge for their modeling. Moreover, the receptive range of most ORs is unknown. The odorant receptor OR5K1 was recently and comprehensively characterized in terms of cognate agonists. Here, we report two additional agonists and functional data of the most potent compound on two mutants, L1043.32 and L2556.51. Experimental data was used to guide the investigation of the binding modes of OR5K1 ligands into the orthosteric binding site using structural information from AI-driven modeling, as recently released in the AlphaFold Protein Structure Database, and from homology modeling. Induced-fit docking simulations were used to sample the binding site conformational space for ensemble docking. Mutagenesis data guided side chain residue sampling and model selection. We obtained models that could better rationalize the different activity of active (agonist) versus inactive molecules with respect to starting models and also capture differences in activity related to minor structural differences. Therefore, we provide a model refinement protocol that can be applied to model the orthosteric binding site of ORs as well as that of GPCRs with low sequence identity to available templates.

Heterodimerization of Chemoreceptors TAS1R3 and mGlu2 in Human Blood Leukocytes.

The expression of canonical chemosensory receptors of the tongue, such as the heteromeric sweet taste (TAS1R2/TAS1R3) and umami taste (TAS1R1/TAS1R3) receptors, has been demonstrated in many extra-oral cells and tissues. Gene expression studies have revealed transcripts for all TAS1 and metabotropic glutamate (mGlu) receptors in different types of immune cells, where they are involved, for example, in the chemotaxis of human neutrophils and the protection of T cells from activation-induced cell death. Like other class-C G protein-coupling receptors (GPCRs), TAS1Rs and mGlu receptors form heteromers within their families. Since mGlu receptors and TAS1R1/TAS1R3 share the same ligand, monosodium glutamate (MSG), we hypothesized their hitherto unknown heteromerization across receptor families in leukocytes. Here we show, by means of immunocytochemistry and co-IP/Western analysis, that across class-C GPCR families, mGlu2 and TAS1R3 co-localize and heterodimerize in blood leukocytes. Expressing the recombinant receptors in HEK-293 cells, we validated their heterodimerization by bioluminescence resonance energy transfer. We demonstrate MSG-induced, mGlu2/TAS1R3 heteromer-dependent gain-of-function and pertussis toxin-sensitive signaling in luminescence assays. Notably, we show that mGlu2/TAS1R3 is necessary and sufficient for MSG-induced facilitation of N-formyl-methionyl-leucyl-phenylalanine-stimulated IL-8 secretion in neutrophils, using receptor-specific antagonists. In summary, our results demonstrate mGlu2/TAS1R3 heterodimerization in leukocytes, suggesting cellular function-tailored chemoreceptor combinations to modulate cellular immune responses.

Conserved C-terminal motifs in odorant receptors instruct their cell surface expression and cAMP signaling.

The highly individual plasma membrane expression and cAMP signaling of odorant receptors have hampered their ligand assignment and functional characterization in test cell systems. Chaperones have been identified to support the cell surface expression of only a portion of odorant receptors, with mechanisms remaining unclear. The presence of amino acid motifs that might be responsible for odorant receptors' individual intracellular retention or cell surface expression, and thus, for cAMP signaling, is under debate: so far, no such protein motifs have been suggested. Here, we demonstrate the existence of highly conserved C-terminal amino acid motifs, which discriminate at least between class-I and class-II odorant receptors, with their numbers of motifs increasing during evolution, by comparing C-terminal protein sequences from 4808 receptors across eight species. Truncation experiments and mutation analysis of C-terminal motifs, largely overlapping with helix 8, revealed single amino acids and their combinations to have differential impact on the cell surface expression and on stimulus-dependent cAMP signaling of odorant receptors in NxG 108CC15 cells. Our results demonstrate class-specific and individual C-terminal motif equipment of odorant receptors, which instruct their functional expression in a test cell system, and in situ may regulate their individual cell surface expression and intracellular cAMP signaling.

An evolutionary conserved olfactory receptor for foodborne and semiochemical alkylpyrazines.

Molecular recognition is a fundamental principle in biological systems. The olfactory detection of both food and predators via ecological relevant odorant cues are abilities of eminent evolutionary significance for many species. Pyrazines are such volatile cues, some of which act as both human-centered key food odorants (KFOs) and semiochemicals. A pyrazine-selective odorant receptor has been elusive. Here we screened 2,3,5-trimethylpyrazine, a KFO and semiochemical, and 2,5-dihydro-2,4,5-trimethylthiazoline, an innate fear-associated non-KFO, against 616 human odorant receptor variants, in a cell-based luminescence assay. OR5K1 emerged as sole responding receptor. Tested against a comprehensive collection of 178 KFOs, we newly identified 18 pyrazines and (2R/2S)-4-methoxy-2,5-dimethylfuran-3(2H)-one as agonists. Notably, OR5K1 orthologs in mouse and domesticated species displayed a human-like, potency-ranked activation pattern of pyrazines, suggesting a domestication-led co-evolution of OR5K1 and its orthologs. In summary, OR5K1 is a specialized olfactory receptor across mammals for the detection of pyrazine-based key food odors and semiochemicals.

Copper-mediated thiol potentiation and mutagenesis-guided modeling suggest a highly conserved copper-binding motif in human OR2M3.

Sulfur-containing compounds within a physiological relevant, natural odor space, such as the key food odorants, typically constitute the group of volatiles with the lowest odor thresholds. The observation that certain metals, such as copper, potentiate the smell of sulfur-containing, metal-coordinating odorants led to the hypothesis that their cognate receptors are metalloproteins. However, experimental evidence is sparse-so far, only one human odorant receptor, OR2T11, and a few mouse receptors, have been reported to be activated by sulfur-containing odorants in a copper-dependent way, while the activation of other receptors by sulfur-containing odorants did not depend on the presence of metals. Here we identified an evolutionary conserved putative copper interaction motif CC/CSSH, comprising two copper-binding sites in TMH5 and TMH6, together with the binding pocket for 3-mercapto-2-methylpentan-1-ol in the narrowly tuned human receptor OR2M3. To characterize the copper-binding motif, we combined homology modeling, docking studies, site-directed mutagenesis, and functional expression of recombinant ORs in a cell-based, real-time luminescence assay. Ligand activation of OR2M3 was potentiated in the presence of copper. This effect of copper was mimicked by ionic and colloidal silver. In two broadly tuned receptors, OR1A1 and OR2W1, which did not reveal a putative copper interaction motif, activation by their most potent, sulfur-containing key food odorants did not depend on the presence of copper. Our results suggest a highly conserved putative copper-binding motif to be necessary for a copper-modulated and thiol-specific function of members from three subfamilies of family 2 ORs.

Beyond the Flavour: The Potential Druggability of Chemosensory G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) belong to the largest class of drug targets. Approximately half of the members of the human GPCR superfamily are chemosensory receptors, including odorant receptors (ORs), trace amine-associated receptors (TAARs), bitter taste receptors (TAS2Rs), sweet and umami taste receptors (TAS1Rs). Interestingly, these chemosensory GPCRs (csGPCRs) are expressed in several tissues of the body where they are supposed to play a role in biological functions other than chemosensation. Despite their abundance and physiological/pathological relevance, the druggability of csGPCRs has been suggested but not fully characterized. Here, we aim to explore the potential of targeting csGPCRs to treat diseases by reviewing the current knowledge of csGPCRs expressed throughout the body and by analysing the chemical space and the drug-likeness of flavour molecules.

Structural determinants of a conserved enantiomer-selective carvone binding pocket in the human odorant receptor OR1A1.

Chirality is a common phenomenon within odorants. Most pairs of enantiomers show only moderate differences in odor quality. One example for enantiomers that are easily discriminated by their odor quality is the carvones: humans significantly distinguish between the spearmint-like (R)-(-)-carvone and caraway-like (S)-(+)-carvone enantiomers. Moreover, for the (R)-(-)-carvone, an anosmia is observed in about 8% of the population, suggesting enantioselective odorant receptors (ORs). With only about 15% de-orphaned human ORs, the lack of OR crystal structures, and few comprehensive studies combining in silico and experimental approaches to elucidate structure-function relations of ORs, knowledge on cognate odorant/OR interactions is still sparse. An adjusted homology modeling approach considering OR-specific proline-caused conformations, odorant docking studies, single-nucleotide polymorphism (SNP) analysis, site-directed mutagenesis, and subsequent functional studies with recombinant ORs in a cell-based, real-time luminescence assay revealed 11 amino acid positions to constitute an enantioselective binding pocket necessary for a carvone function in human OR1A1 and murine Olfr43, respectively. Here, we identified enantioselective molecular determinants in both ORs that discriminate between minty and caraway odor. Comparison with orthologs from 36 mammalian species demonstrated a hominid-specific carvone binding pocket with about 100% conservation. Moreover, we identified loss-of-function SNPs associated with the carvone binding pocket of OR1A1. Given carvone enantiomer-specific receptor activation patterns including OR1A1, our data suggest OR1A1 as a candidate receptor for constituting a carvone enantioselective phenotype, which may help to explain mechanisms underlying a (R)-(-)-carvone-specific anosmia in humans.

The Broadly Tuned Odorant Receptor OR1A1 is Highly Selective for 3-Methyl-2,4-nonanedione, a Key Food Odorant in Aged Wines, Tea, and Other Foods.

Key food odorants are the most relevant determinants by which we detect, recognize, and hedonically evaluate the aroma of foods and beverages. Odorants are detected by our chemical sense of olfaction, comprising a set of approximately 400 different odorant receptor types. However, the specific receptor activity patterns representing the aroma percepts of foods or beverages, as well as the key food odorant agonist profiles of single-odorant receptors, are largely unknown. We aimed to establish comprehensive key food odorant agonist profiles of 2 unrelated, broadly tuned receptors, OR1A1 and OR2W1, that had been associated thus far with mostly non-key food odorants and shared some of these agonists. By screening both receptors against 190 key food odorants in a cell-based luminescence assay, we identified 14 and 18 new key food odorant agonists for OR1A1 and OR2W1, respectively, with 3-methyl-2,4-nonanedione emerging as the most potent agonist for OR1A1 by 3 orders of magnitude, with a submicromolar half maximal effective concentration. 3-Methyl-2,4-nonanedione has been associated with a prune note in oxidized wine and is an aroma determinant in tea and apricots. Further screening against the entire set of 391 human odorant receptors revealed that 30 or 300 µmol/L 3-methyl-2,4-nonanedione activated only 1 receptor, OR1A1, suggesting a unique role of OR1A1 for the most sensitive detection of this key food odorant in wine, tea, and other food matrices.

OR2M3: A Highly Specific and Narrowly Tuned Human Odorant Receptor for the Sensitive Detection of Onion Key Food Odorant 3-Mercapto-2-methylpentan-1-ol.

The detection of key food odorants appears to be an important capability of odorant receptors. Here, thiols occupy an outstanding position among the 230 known key food odorants because of their very low odor thresholds. Members of the homologous series of 3-mercapto-2-methylalkan-1-ols have been described as onion key food odorants or food constituents and are detected at logarithmically different thresholds. 3-Mercapto-2-methylpentan-1-ol being the only key food odorant within this series also has the lowest odor threshold. Most odorants typically activate combinations of odorant receptors, which may be narrowly or broadly tuned. Consequently, a specific receptor activation pattern will define an odor quality. In contrast, here we show that just 1 of the 391 human odorant receptors, OR2M3, responded exclusively to 3-mercapto-2-methylpentan-1-ol of the 190 key food odorants tested, with a half maximal effective concentration at submicromolar concentration. Moreover, neither the Denisovan OR2M3 nor the closest OR2M3 homologs from five species did respond to this compound. This outstanding specificity of extremely narrowly tuned human OR2M3 can explain both odor qualities and odor threshold trend within a homologous series of 3-mercapto-2-methylalkan-1-ols and suggests a modern human-specific, food-related function of OR2M3 in detecting a single onion key food odorant.

TRPC6 G757D Loss-of-Function Mutation Associates with FSGS.

FSGS is a CKD with heavy proteinuria that eventually progresses to ESRD. Hereditary forms of FSGS have been linked to mutations in the transient receptor potential cation channel, subfamily C, member 6 (TRPC6) gene encoding a nonselective cation channel. Most of these TRPC6 mutations cause a gain-of-function phenotype, leading to calcium-triggered podocyte cell death, but the underlying molecular mechanisms are unclear. We studied the molecular effect of disease-related mutations using tridimensional in silico modeling of tetrameric TRPC6. Our results indicated that G757 is localized in a domain forming a TRPC6-TRPC6 interface and predicted that the amino acid exchange G757D causes local steric hindrance and disruption of the channel complex. Notably, functional characterization of model interface domain mutants suggested a loss-of-function phenotype. We then characterized 19 human FSGS-related TRPC6 mutations, the majority of which caused gain-of-function mutations. However, five mutations (N125S, L395A, G757D, L780P, and R895L) caused a loss-of-function phenotype. Coexpression of wild-type TRPC6 and TRPC6 G757D, mimicking heterozygosity observed in patients, revealed a dominant negative effect of TRPC6 G757D. Our comprehensive analysis of human disease-causing TRPC6 mutations reveals loss of TRPC6 function as an additional concept of hereditary FSGS and provides molecular insights into the mechanism responsible for the loss-of-function phenotype of TRPC6 G757D in humans.

Nature's chemical signatures in human olfaction: a foodborne perspective for future biotechnology.

The biocatalytic production of flavor naturals that determine chemosensory percepts of foods and beverages is an ever challenging target for academic and industrial research. Advances in chemical trace analysis and post-genomic progress at the chemistry-biology interface revealed odor qualities of nature's chemosensory entities to be defined by odorant-induced olfactory receptor activity patterns. Beyond traditional views, this review and meta-analysis now shows characteristic ratios of only about 3 to 40 genuine key odorants for each food, from a group of about 230 out of circa 10 000 food volatiles. This suggests the foodborn stimulus space has co-evolved with, and roughly match our circa 400 olfactory receptors as best natural agonists. This perspective gives insight into nature's chemical signatures of smell, provides the chemical odor codes of more than 220 food samples, and beyond addresses industrial implications for producing recombinants that fully reconstruct the natural odor signatures for use in flavors and fragrances, fully immersive interactive virtual environments, or humanoid bioelectronic noses.

Petrol Note in Riesling - 1,1,6-Trimethyl-1,2-dihydronaphthalene (TDN) Selectively Activates Human Odorant Receptor OR8H1.

Grapevine (Vitis vinifera) is one of the most important perennial fruit plants. The variety Riesling stands out by developing a characteristic petrol-like odor note during aging, elicited by the aroma compound 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN). The UV-dependent TDN contents differ largely among Rieslings grown in the northern versus the southern hemisphere. Highest TDN concentrations were found in Australian Rieslings, where TDN is a scoring ingredient. In contrast, in Rieslings from Europe, for example, TDN may be a tending cause of rejection. A human receptor for TDN has been unknown. Here, we report on the identification of OR8H1 as a TDN-selective odorant receptor, out of a library of 766 odorant receptor variants. OR8H1 is selectively tuned to six carbon ring structures, identified by screening a collection of 180 key food odorants, using a HEK-293 cell-based cAMP luminescence assay equipped with the GloSensor technology.

Geosmin, a Food- and Water-Deteriorating Sesquiterpenoid and Ambivalent Semiochemical, Activates Evolutionary Conserved Receptor OR11A1.

Geosmin, a ubiquitous volatile sesquiterpenoid of microbiological origin, is causative for deteriorating the quality of many foods, beverages, and drinking water, by eliciting an undesirable "earthy/musty" off-flavor. Moreover, and across species from worm to human, geosmin is a volatile, chemosensory trigger of both avoidance and attraction behaviors, suggesting its role as semiochemical. Volatiles typically are detected by chemosensory receptors of the nose, which have evolved to best detect ecologically relevant food-related odorants and semiochemicals. An insect receptor for geosmin was recently identified in flies. A human geosmin-selective receptor, however, has been elusive. Here, we report on the identification and characterization of a human odorant receptor for geosmin, with its function being conserved in orthologs across six mammalian species. Notably, the receptor from the desert-dwelling kangaroo rat showed a more than 100-fold higher sensitivity compared to its human ortholog and detected geosmin at low nmol/L concentrations in extracts from geosmin-producing actinomycetes.

Impaired metal perception and regulation of associated human foliate papillae tongue transcriptome in long-COVID-19.

Chemosensory impairment is an outstanding symptom of SARS-CoV-2 infections. We hypothesized that measured sensory impairments are accompanied by transcriptomic changes in the foliate papillae area of the tongue. Hospital personnel with known SARS-CoV-2 immunoglobulin G (IgG) status completed questionnaires on sensory perception (n = 158). A subcohort of n = 141 participated in forced choice taste tests, and n = 43 participants consented to donate tongue swabs of the foliate papillae area for whole transcriptome analysis. The study included four groups of participants differing in IgG levels (≥ 10 AU/mL = IgG+; < 10 AU/mL = IgG-) and self-reported sensory impairment (SSI±). IgG+ subjects not detecting metallic taste had higher IgG+ levels than IgG+ participants detecting iron gluconate (p = 0.03). Smell perception was the most impaired biological process in the transcriptome data from IgG+/SSI+ participants subjected to gene ontology enrichment. IgG+/SSI+ subjects demonstrated lower expression levels of 166 olfactory receptors (OR) and 9 taste associated receptors (TAS) of which OR1A2, OR2J2, OR1A1, OR5K1 and OR1G1, as well as TAS2R7 are linked to metallic perception. The question raised by this study is whether odorant receptors on the tongue (i) might play a role in metal sensation, and (ii) are potential targets for virus-initiated sensory impairments, which needs to be investigated in future functional studies.

The human TAS2R16 receptor mediates bitter taste in response to beta-glucopyranosides.

Bitter taste generally causes aversion, which protects humans from ingesting toxic substances. But bitter flavors also contribute to the palatability of food and beverages, thereby influencing nutritional habits in humans. Although many studies have examined bitter taste, the underlying receptor mechanisms remain poorly understood. Anatomical, functional and genetic data from rodents suggest the existence of a family of receptors that are responsive to bitter compounds. Here we report that a human member of this family, TAS2R16, is present in taste receptor cells on the tongue and is activated by bitter beta-glucopyranosides. Responses to these phytonutrients show a similar concentration dependence and desensitization in transfected cells and in experiments assessing taste perception in humans. Bitter compounds consisting of a hydrophobic residue attached to glucose by a beta-glycosidic bond activate TAS2R16. Thus, TAS2R16 links the recognition of a specific chemical structure to the perception of bitter taste. If the ability of TAS2R16 to detect substances with common molecular properties is typical of the bitter receptor family, it may explain how a few receptors permit the perception of numerous bitter substances.

[6]-Gingerol Facilitates CXCL8 Secretion and ROS Production in Primary Human Neutrophils by Targeting the TRPV1 Channel.

SCOPE: Clarifying the function of sensory active TRP (transient receptor potential) channels in non-sensory tissue is of growing interest, especially with regard to food ingredients in nutritionally relevant concentrations. The study hypothesizes the TRPV1 agonist [6]-gingerol to facilitate cellular immune responses of primary human neutrophils, after treatment with 50 nM, a concentration that can be reached in the circulation after habitual dietary intake. METHODS AND RESULTS: qRT-PCR analyses reveal a high abundancy of TRP channel RNA expression in the types of primary leukocytes investigated, namely neutrophils, monocytes, NK cells, T cells, and B cells. Incubation of neutrophils with 50 nM of the known TRPV1 ligand [6]-gingerol led to increased surface expression of CD11b, CD66b, and the fMLF receptor FPR1, as shown by flow cytometry. Upon subsequent stimulation with fMLF, the neutrophils display an about 30% (p < 0.05) increase in CXCL8 secretion as well as in ROS production. Pharmacological inhibition of TRPV1 by trans-tert-butylcyclohexanol abolishes the [6]-gingerol induced effects. CONCLUSIONS: The TRPV1 channel is functionally expressed in human neutrophils. Activation of the channel with [6]-gingerol as a food-derived ligand in nutritionally relevant concentrations leads to an enhanced responsiveness in the cells towards activating stimuli, thereby facilitating a canonical cellular immune response in human neutrophils.

The multi-faceted food odorant 4-methylphenol selectively activates evolutionary conserved receptor OR9Q2.

4-Methylphenol is a food-related odor-active volatile with a high recognition factor, due to its horse stable-like, fecal odor quality. Its ambivalent hedonic impact as key aroma compound, malodor, and semiochemical has spurred the search for its cognate, chemosensory odorant receptors across species. A human odorant receptor for the highly characteristic 4-methylphenol has been elusive. Here, we identified and characterized human receptor OR9Q2 to be tuned to purified 4-methylphenol, but not to its contaminant isomer 3-methylphenol. This highly selective function of OR9Q2 complements an exclusive phenol detection gap in the ancient, most broadly tuned human odorant receptor OR2W1. Moreover, a 4-methylphenol function is evolutionary conserved in phylogenetically related OR9Q2 orthologs from chimpanzee, mouse, and cow. Notably, the cow receptor outperformed human OR9Q2 10-fold in signal strength, consonant with previous reports of 4-methylphenol as a bovine pheromone. Our results suggest OR9Q2 as best sensor for the key food odorant, malodor, and semiochemical 4-methylphenol.

Sweetener System Intervention Shifted Neutrophils from Homeostasis to Priming.

BACKGROUND: Non-nutritive sweeteners (NNS) are part of personalized nutrition strategies supporting healthy glycemic control. In contrast, the consumption of non-nutritive sweeteners has been related to person-specific and microbiome-dependent glycemic impairments. Reports on the effects of NNS on our highly individual cellular immune system are sparse. The recent identification of taste receptor expression in a variety of immune cells, however, suggested their immune-modulatory relevance. METHODS: We studied the influence of a beverage-typical NNS system on the transcriptional profiling of sweetener-cognate taste receptors, selected cytokines and their receptors, and on Ca2+ signaling in isolated blood neutrophils. We determined plasma concentrations of saccharin, acesulfame-K, and cyclamate by HPLC-MS/MS, upon ingestion of a soft drink-typical sweetener surrogate. In an open-labeled, randomized intervention study, we determined pre- versus post-intervention transcript levels by RT-qPCR of sweetener-cognate taste receptors and immune factors. RESULTS: Here we show that the consumption of a food-typical sweetener system modulated the gene expression of cognate taste receptors and induced the transcriptional regulation signatures of early homeostasis- and late receptor/signaling- and inflammation-related genes in blood neutrophils, shifting their transcriptional profile from homeostasis to priming. Notably, sweeteners at postprandial plasma concentrations facilitated fMLF (N-formyl-Met-Leu-Phe)-induced Ca2+ signaling. CONCLUSIONS: Our results support the notion of sweeteners priming neutrophils to higher alertness towards their adequate stimuli.

The key food odorant receptive range of broadly tuned receptor OR2W1.

Mammals perceive a multitude of odorants by their chemical sense of olfaction, a high-dimensional stimulus-detection system, with hundreds of narrowly or broadly tuned receptors, enabling pattern recognition by the brain. Cognate receptor-agonist information, however, is sparse, and the role of broadly tuned odorant receptors for encoding odor quality remains elusive. Here, we screened IL-6-HaloTag®-OR2W1 and haplotypes against 187 out of 230 defined key food odorants using the GloSensor™ system in HEK-293 cells, yielding 48 new agonists. Altogether, key food odorants represent about two-thirds of now 153 reported agonists of OR2W1, the highest number of agonists known for a mammalian odorant receptor. In summary, we characterized OR2W1 as a human odorant receptor, with a chemically diverse but exclusive receptive range, complementary to chemical subgroups covered by evolutionary younger, highly selective receptors. Our data suggest OR2W1 to be suited for participating in the detection of many foodborne odorants.

Metabolites of Key Flavor Compound 2,3,5-Trimethylpyrazine in Human Urine.

Pyrazines are among the most important compound class conveying the odor impressions "roasty", "nutty", and "earthy". They are formed by the Maillard reaction and occur ubiquitously in heated foods. The excretion of metabolites of the key flavor odorant 2,3,5-trimethylpyrazine, abundant in the volatile fraction of roasted coffee, was investigated. Based on literature suggestions, putative phase 1 and phase 2 metabolites were synthesized, characterized by nuclear magnetic resonance and mass spectroscopy data and used as standards for targeted, quantitative analysis of coffee drinkers' urine using stable-isotope-dilution-ultrahigh-performance liquid chromatography tandem mass spectroscopy (SIDA-UHPLC-MS/MS). The analysis of spot urine samples from a coffee intervention study revealed 3,6-dimethylpyrazine-2-carboxylic acid, 3,5-dimethylpyrazine-2-carboxylic acid, and 5,6-dimethylpyrazine-2-carboxylic acid were quantitatively dominating metabolites. Only negligible traces of pyrazinemethanols (3,6-dimethyl-2-pyrazinemethanol and 3,5,6-trimethylpyrazine-2-ol), glucuronides ((3,6-dimethylpyrazine-2-yl-)methyl-O-ß-D-glucuronide and (3,5-dimethylpyrazine-2-yl-)methyl-O-ß-D-glucuronide), and sulfates ((3,6-dimethylpyrazine-2-yl-)methyl-sulfate and (3,5-dimethylpyrazine-2-yl-)methyl-sulfate) were detected.